S. uberis is an important cause of mastitis in dairy cattle and is responsible for about 20% of all clinical cases of mastitis (Bramley, A. J. and Dodd, F. H. (1984) J. Dairy Res. 51:481-512; Bramley, A. J. (1987) Animal Health Nutrition 42:12-16; Watts, J. L. (1988) J. Dairy Sci. 71:1616-1624). Since antimicrobial treatment is generally ineffective in treating S. uberis mastitis, the development of control measures must be based on an understanding of virulence factors and protective antigens involved in invasion and protection of the mammary gland (Collins et al. (1988) J. Dairy Res. 55:25-32; Leigh et al. (1990) Res. Vet. Sci. 49:85-87; Marshall et al. (1986) J. Dairy Res. 53: 507-514).
It is known that some S. uberis strains can produce hyaluronic acid capsule (Hill, A. W. (1988) Res. Vet. Sci. 45:400-404), hyaluronidase (Schaufuss et al. (1989) Zentralb. Bakteriol. Ser. A 271:46-53), R-like protein (Groschup, M. H. and Timoney, J. F. (1993) Res. Vet. Sci. 54:124-126), and a cohemolysin, the CAMP factor, also known as UBERIS factor (Skalka, B. and Smola, J. (1981) Zentralb. Bakteriol. Ser. A 249:190-194). However, very little is known of their roles in pathogenicity.
The effect of CAMP factor was first described by Christie et al. in 1944 (Christie et al. (1944) Aus. J. Exp. Biol. Med. Sci. 22:197-200). These authors found that group B streptococci (GBS), such as S. agalactiae, produced a distinct zone of complete hemolysis when grown near the diffusion zone of the Staphylococcus aureus beta-toxin, sphingomyelinase. This phenomenon was called CAMP reaction and the compound for this reaction was characterized as the CAMP factor, an extracellular protein with a molecular weight of 23,500 (Bernheimer et al. (1979) Infect. Immun. 23:838-844). The CAMP factor was subsequently purified from S. agalactiae and characterized as a 25,000 Da protein with a pI of 8.9 (Jürgens et al. (1985) J. Chrom. 348:363-370). The amino acid sequence of S. agalactiae CAMP factor was determined by Rühlmann et al. (Rühlmann et al. (1988) FEBS Lett 235:262-266).
The mechanism of the CAMP reaction has been described. See, e.g., Bernheimer et al. (1979) Infect. Immun. 23:838-844; Sterzik et al. “Interaction of the CAMP factor from S. agalactiae with artificial membranes.” In: Alouf et al., eds. Bacterial protein toxins, London: Academic Press Inc, 1984; 195-196; Sterzik et al. (1985) Zentralbl. Bakteriol. Mikrobiol. Hyg. Abt. 1 Suppl. 15:101-108; Fehrenbach et al. “Role of CAMP-factor (protein B) for virulence.” In: Fehrenbach et al., eds. Bacterial protein toxins, Stuttgart: Gustav Fischer Verlag, 1988; 351-357; Fehrenbach et al. “Interaction of amphiphilic bacterial polypeptides with artificial membranes.” In: Alouf et al., eds. Bacterial protein toxins, London: Academic Press Inc., 1984:317-324.
CAMP factor has lytic action on a variety of target cells including sheep and bovine erythrocytes, as well as on artificial membranes in which membrane phospholipids and sphingomyelin have been hydrolyzed by phospholipase or sphingomyelinase.
The role of CAMP factor in pathogenicity is unclear. A partially purified CAMP factor from S. agalactiae has been shown to be lethal to rabbits when injected intravenously (Skalka, B. and Smola, J. (1981) Zentralbl. Bakteriol. Ser. A 249:190-194). Furthermore, intraperitoneal injection of purified CAMP factor into mice has been shown to significantly raise the pathogenicity of a sublethal dose of group B streptococci (Fehrenbach et al. “Role of CAMP-factor (protein B) for virulence.” In: Fehrenbach et al., eds. Bacterial protein toxins, Stuttgart: Gustav Fischer Verlag, 1988; 351-357). Additionally, like protein A of S. aureus, GBS CAMP factor can bind the Fc sites of immunoglobulins and has therefore been designated protein B (Jürgens et al. (1987) J. Exp. Med. 165:720-732).
In addition to GBS and S. uberis, other bacteria, including Listeria monocytogenes and Listeria seeligeri (Rocourt, J. and Grimont, P. A. D. (1983) Int. J. Syst. Bacteriol. 33:866-869) Aeromonas sp. (Figura, N. and Guglielmetti, P. (1987) J. Clin. Microbiol. 25:1341-1342), Rhodococcus equi (Fraser, G. (1961) Nature 189:246), and certain Vibrio spp. (Kohler, W. (1988) Zentralbl. Bakteriol. Mikrobiol. Hyg. Ser. A 270:35-40) produce reactions similar to the CAMP effect.
The CAMP factor genes of GBS and A. pleuropneumoniae have been cloned and expressed in Escherichia coli (Schneewind et al. (1988) Infect. Immun. 56:2174-2179; Frey et al. (1989) Infect. Immun. 57:2050-2056). Additionally, the gene encoding the CAMP factor from a group A streptococci (GAS) strain, S. pyogenes, has also been isolated (Gase et al. (1999) Infect. Immun. 67:4725-4731). The CAMP protein products were of similar size and possessed homology to the CAMP proteins of S. agalactiae and S. uberis. Antibodies raised against the cloned CAMP protein of A. pleuropneumoniae neutralized the CAMP reaction mediated by the E. coli strain containing the cloned CAMP gene as well as that of A. pleuropneumoniae, and also cross-reacted with the S. agalactiae CAMP factor. In the GAS strains, the distribution of the cfa (CAMP) gene was analyzed. This gene was widely spread among GAS: 82 of 100 clinical GAS isolates produced a positive CAMP reaction. Of the CAMP-negative strains, 17 of the 18 GAS strains contained the cfa gene. Additionally, CAMP activity was detected in streptococci from serogroups C, M, P, R, and U (Gase et al. (1999) Infect. Immun. 67:4725-4731).
However, prior to the present inventors' discovery, the CAMP factor gene of S. uberis had not been cloned. Furthermore, the protective capability of CAMP factor had not been previously studied. Additionally, the production and use of chimeric CAMP factor constructs, containing epitopes derived from CAMP factors from more than one microbe, has not previously been described.